1. Field of the Invention
This invention broadly relates to an improvement in methods for detecting and especially for quantifying the presence of a target molecule, such as an antigen, an antibody or a polynucleotide, in a sample. More particularly, the invention relates to an improved method for conducting an immunoassay or a polynucleotide hybridization assay, which method uses alkaline phosphatase as the reporter enzyme and a reduction of a tetrazolium salt to a formazan as the detection/signaling system for detecting or quantifying a target molecule such as may be present in a biological sample.
2. Description of Related Art
The analysis and detection of minute quantities of substances in biological and non-biological samples, both qualitative and quantitative, has become a routine practice in clinical, diagnostic and analytical laboratories. The most prevalent detection techniques are based on ligand-receptor interactions (e.g., immunoassay-based techniques) and polynucleotide hybridizations.
Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody with an antigen complementary to it. Such assays have been used to detect antibodies produced in response to infection, components of pathological agents, levels of drugs, hormones, enzymes, etc. In addition to medicinal applications, immunoassays also have been applied in the manufacturing industries, for example, for the detection of food contaminants.
In one approach, commonly referred to as a sandwich assay, a sample to be tested for the presence of a particular target molecule which is one member of an immunological complementary binding pair, is bound to a surface of a solid support. The surface can either be nonimmunological or can harbor an excess of the complement of the desired target molecule (i.e., corresponding antibody or antigen). The surface containing the bound sample then is exposed to an excess of a probe, which also generally functions as a reporter molecule, generally a labeled-antibody or a labeled-antigen reagent (as appropriate), labeled with an enzyme (i.e., an enzyme conjugate), to cause an immunological reaction between the target molecule in the bound sample and the labeled reagent. After removing unreacted labeled reagent or probe from the solid surface, the quantity of bound or unbound labeled reporter molecule is detected or quantified by either contacting the solid surface or the associated liquid phase, respectively, with a detection system containing a signaling reagent, e.g., a substrate for the enzyme that undergoes some detectable change, such as the generation of a colored species, upon reaction with the enzyme.
In a competitive immunoassay, the sample containing an unknown quantity of the target molecule is simultaneously exposed to a solid surface having bound thereto its complementary immunological partner, i.e., bound antigen or bound antibody, with a known amount of a labeled-target molecule. The bound, labeled molecule then is quantitated to determine indirectly the total quantity of target molecule in the sample. At equilibrium, the level of bound labeled target molecule is inversely related to the concentration of the target molecule in the sample.
The reporter molecule (generally either a labeled-antigen or a labeled-antibody) is a heterologous component, usually an enzyme conjugate, which can be detected through a signaling reagent, normally a substrate for the selected enzyme. In the case of an enzyme, a first complex forming site is utilized to attach (usually via a covalent bond) the enzyme to the probe (e.g., to the antigen or antibody, often a polypeptide) and the second (and any additional) complex forming site(s) is(are) utilized to activate the signaling reagent of the detection system, with each complex formed being different and not interfering with each other. The signal thus can be used to demonstrate the presence of the heterologous component and, in turn, the complementary binding partner of the labeled reporter complex (enzyme conjugate).
In a polynucleotide sequence detection assay or polynucleotide hybridization, the non-covalent binding of a labeled polynucleotide sequence or a nucleic acid probe to a complementary sequence of the target molecule is determined under hybridization conditions in accordance with the Watson-Crick base pairing of adenine and thymine, and guanine and cytosine. The nucleic acid probe is modified by a heterologous moiety and the heterologous moiety can be detected through a signaling moiety, in a manner analogous to that described above for immunoassays
Techniques also are known for amplifying the signal produced by such assays, indicative of the presence of a target molecule, using for example multiple ligand-receptor pairs. These and other immunoassay and hybridization techniques are well know to those skilled in the art and all can be used in combination with the present invention.
It is an object of this invention, therefore, to provide a novel method of conducting such ligand-receptor assays (e.g., an immunoassay) and polynucleotide hybridizations.
It is another object of this invention to provide a novel detection/signaling system for indicating or quantitating the presence of a target molecule in a sample.
It is a further object of the invention to provide a detection/signaling system for indicating or quantitating the presence of a target molecule in a sample which uses alkaline phosphatase as the reporter enzyme and a reduction of a tetrazolium salt to a formazan as a coloremetric indicator.
It is yet another object of the present invention to provide a storage stable liquid reagent for use as the detection/signaling reagent in an enzyme-linked immunoassay or a polynucleotide hybridization that has an improved sensitivity at low concentrations of the reporter enzyme.